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Assessing mitochondrial heteroplasmy using next generation sequencing: A note of caution.

ABSTRACT: The mitochondrial genome has recently become the focus of several high-impact next-generation sequencing studies investigating the effect of mutations in disease and assessing the efficacy of mitochondrial replacement therapies. However, these studies have failed to take into consideration the capture of recurring translocations of mitochondrial DNA to the nuclear genome, known as nuclear mitochondrial sequences (NUMTs), continuing to align sequence data to the revised Cambridge reference sequence alone. Here, using different mtDNA enrichment techniques and a variety of tissues, we demonstrate that NUMTs are present in sequence data and that, dependent upon downstream analysis, are at a level which affects variant calling.

SUBMITTER: Santibanez-Koref M

PROVIDER: S-EPMC6509278 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Assessing mitochondrial heteroplasmy using next generation sequencing: A note of caution.

Santibanez-Koref Mauro M Griffin Helen H Turnbull Douglass M DM Chinnery Patrick F PF Herbert Mary M Hudson Gavin G

Mitochondrion 20180809

The mitochondrial genome has recently become the focus of several high-impact next-generation sequencing studies investigating the effect of mutations in disease and assessing the efficacy of mitochondrial replacement therapies. However, these studies have failed to take into consideration the capture of recurring translocations of mitochondrial DNA to the nuclear genome, known as nuclear mitochondrial sequences (NUMTs), continuing to align sequence data to the revised Cambridge reference sequen ...[more]

PMID: 30098421

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Project description:Mitochondrial DNA is a valuable taxonomic marker due to its relatively fast rate of evolution. In Trypanosoma cruzi, the causative agent of Chagas disease, the mitochondrial genome has a unique structural organization consisting of 20-50 maxicircles (?20 kb) and thousands of minicircles (0.5-10 kb). T. cruzi is an early diverging protist displaying remarkable genetic heterogeneity and is recognized as a complex of six discrete typing units (DTUs). The majority of infected humans are asymptomatic for life while 30-35% develop potentially fatal cardiac and/or digestive syndromes. However, the relationship between specific clinical outcomes and T. cruzi genotype remains elusive. The availability of whole genome sequences has driven advances in high resolution genotyping techniques and re-invigorated interest in exploring the diversity present within the various DTUs.To describe intra-DTU diversity, we developed a highly resolutive maxicircle multilocus sequence typing (mtMLST) scheme based on ten gene fragments. A panel of 32 TcI isolates was genotyped using the mtMLST scheme, GPI, mini-exon and 25 microsatellite loci. Comparison of nuclear and mitochondrial data revealed clearly incongruent phylogenetic histories among different geographical populations as well as major DTUs. In parallel, we exploited read depth data, generated by Illumina sequencing of the maxicircle genome from the TcI reference strain Sylvio X10/1, to provide the first evidence of mitochondrial heteroplasmy (heterogeneous mitochondrial genomes in an individual cell) in T. cruzi.mtMLST provides a powerful approach to genotyping at the sub-DTU level. This strategy will facilitate attempts to resolve phenotypic variation in T. cruzi and to address epidemiologically important hypotheses in conjunction with intensive spatio-temporal sampling. The observations of both general and specific incidences of nuclear-mitochondrial phylogenetic incongruence indicate that genetic recombination is geographically widespread and continues to influence the natural population structure of TcI, a conclusion which challenges the traditional paradigm of clonality in T. cruzi.

Project description:BackgroundAccumulating evidence implicates mitochondrial DNA (mtDNA) alleles, which are independent of the nuclear genome, in disease, especially in human metabolic diseases. However, this area of investigation has lagged behind in researching the nuclear alleles in complex traits, for example, in gout.MethodsNext-generation sequencing was utilized to investigate the relationship between mtDNA alleles and phenotypic variations in 52 male patients with gout and 104 age-matched male non-gout controls from the Taiwan Biobank whole-genome sequencing samples. Differences from a reference sequence (GRCh38) were identified. The sequence kernel association test (SKAT) was applied to identify gout-associated alleles in mitochondrial genes. The tools Polymorphism Phenotyping, Sorting Intolerant From Tolerant (SIFT), Predict the pathology of Mutations (PMUT), Human Mitochondrial Genome Database (mtDB), Multiple Alignment using Fast Fourier Transform (MAFFT), and Mammalian Mitochondrial tRNA Genes (Mamit-tRNA) were used to evaluate pathogenicity of alleles. Validation of selected alleles by quantitative polymerase chain reaction of single nucleotide polymorphisms (qPCR SNPs) was also performed.ResultsWe identified 456 alleles in patients with gout and 640 alleles in non-gout controls with 274 alleles shared by both. Mitochondrial genes were associated with gout, with MT-CO3, MT-TA, MT-TC, and MT-TT containing potentially pathogenic gout-associated alleles and displaying evidence of gene-gene interactions. All heteroplasmy levels of potentially pathogenic alleles exceeded metabolic thresholds for pathogenicity. Validation assays confirmed the next-generation sequencing results of selected alleles. Among them, potentially pathogenic MT-CO3 alleles correlated with high-density lipoprotein (HDL) levels (P?=?0.034).ConclusionThis study provided two scientific insights. First, this was the most extensive mitochondrial genomic profiling associated with gout. Second, our results supported the roles of mitochondria in gout and HDL, and this comprehensive analysis framework can be applied to other diseases in which mitochondrial dysfunction has been implicated.

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Assessing mitochondrial heteroplasmy using next generation sequencing: A note of caution.

Publications

Assessing mitochondrial heteroplasmy using next generation sequencing: A note of caution.

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